Studies of DNA and proteins in biological laboratories often involve the fluorescence imaging of planar samples of moderate to large area, such as those ranging from 5 cm×5 cm up to 26 cm×26 cm. Common examples of these samples are electrophoresis gels and blotting membranes. The imaging is often performed by camera-based instruments that include an excitation light source of different wavelengths, depending on the sample matrix and the species sought to be detected within the matrix. Gels for example are substantially transparent to ultraviolet (UV), visible, and near infrared (NIR) light, and can be detected from above with illumination from below in the “trans” mode, i.e., by a transilluminator. Transillumination is typically performed with UV-B light in conjunction with a filter that blocks visible-range wavelengths and transmits UV wavelengths, or with phosphor light conversion plates, plastic light conversion plates, or both, to convert the UV light into visible light. Examples of these plates are the XCITABLUE™ Conversion Screen of Bio-Rad Laboratories, Inc. (Hercules, Calif., USA), and the UV/White Light Conversion Screen of UVP, LLC (Upland, Calif., USA). Gels can also be illuminated from above, i.e., on the same side where detection is performed, in the “epi” mode. Epi-illumination is typically performed with an optically filtered light source, which can be either a bulb or an LED. Blot samples for example are optically opaque in the UV, visible, and NIR ranges and must be epi-illuminated.
Epi-illumination imaging systems are sold by Bio-Rad Laboratories, Inc., Fuji Manufacturing USA, Inc. (Greenwood, S.C., USA), GE Healthcare Bio-Sciences, Inc. (Piscataway, N.J., USA), Syngene (Frederick, Md., USA), and ProteinSimple (Santa Clara, Calif., USA). While both light-emitting diodes (LEDs) and bulbs can be used, LEDs are highly favored since they offer high spectral purity and intensity. The illumination produced by LED illuminators is highly non-uniform illumination, however, with a typical radial reduction in intensity of 90% or more from the center of the sample to the edge. This presents a challenge, because non-uniform sample illumination results in a correspondingly non-uniform sample signal which must be made uniform by flat fielding using calibration samples and software. Flat fielding is also used to remove the effects of lens roll-off and optical filter roll-off, both with angle. A single technique such as flat fielding is not well equipped to address all three effects—illumination non-uniformity, lens roll-off, and optical filter roll-off. What is needed therefore is a means of achieving highly uniform illumination, leaving lens and filter roll-off issues to flat fielding which more effectively addresses these issues.